Provisional extracellular matrix citrullination as a stimulus for pathological fibroblast activation

Abstract

Activated fibroblasts have been implicated as drivers of disease progression in a variety of conditions including rheumatoid arthritis (RA), fibrotic diseases, and cancer, and importantly, there currently exists a dearth of understanding regarding how fibroblasts become activated. The central hypothesis of this work is that an inflammation-mediated provisional extracellular matrix (pECM) modification called citrullination may be responsible in whole or in part for activated fibroblast phenotypes. In Aim 1 the influence of citrullinated fibronectin (Cit Fn) was investigated at a molecular scale utilizing a combination of mass spectrometry (MS), bio-layer interferometry (BLI), and immunocytochemistry (ICC) and force-inducible co-immunoprecipitation methods to probe for integrin interactions and downstream signaling. A total of 24 unique citrullination sites were discovered that effectively cause a shift in integrin preference from αvβ3 towards α5β1 that ultimately leads to enhancement of mechanotransduction signaling. In Aim 2 the ability of citrullinated pECM (Cit-pECM) as a stand-alone stimulus to influence fibroblast behavior was explored through a variety of adhesion, migration, apoptosis, proliferation, contraction, and focal adhesion (FA) turnover assays, in addition to atomic force microscopy (AFM). Results indicate that Cit-pECM is sufficient to impact several aspects of fibroblast behavior including increasing FA turnover which leads to an enhancement of cell migration. Overall these findings signify that Cit-pECM is influential in the context fibroblast activation and it may therefore constitute a promising therapeutic target in the treatment of a variety of inflammatory conditions.